In 1990, G. J. Puppels et al., reported their invention of laser confocal Raman microscopy technology in the journal Nature, which combined Raman spectrum detection technology and laser confocal microscopy technology, and which was a revolutionary breakthrough in Raman technology. This technology not only came into the high resolution chromatography imaging characteristics of confocal microscopy, but also could perform spectral analysis to samples; which can implement chromatography detection with high resolution for the micro-area spectrum of the sample. These remarkable advantages make the laser confocal Raman microscopy technology out of common in the field of spectral measurement and rapidly develop into an important means of structure and composition analysis of sample. The laser confocal Raman microscopy technology can be widely used in the forward basic research of many disciplines, such as chemistry, biology, medicine, physics, geology, collection of evidences, forensic science and etc.
Currently, the principle of a typical laser confocal Raman microscopy is shown in FIG. 2. After sequentially passing through a first converging lens, a first pinhole, an eighth converging lens, a first beam splitting system, a quarter wave plate and an objective lens along the light path, the laser focuses on a measured sample and excites the Raman scattering light carrying the spectral characteristics of the sample; the sample is moved, in such a manner that the Raman scattering light corresponding to different parts of the tested sample passes through the quarter wave plate again and is reflected by the first beam splitting system, and then through a fourth converging lens, a fourth pinhole and a fifth converging lens, focuses into a first spectrometer to detect spectrum.
The rapid development of modern science and technology has come up with an improved requirement to the detection capacities of the micro-area spectrum and the spatial resolution; if the spatial resolution is desired to be improved, the system should be focalized accurately. In optical detection system, the measuring converging spot has a minimum size and a strongest intensity of excitation light when it is in focus. Therefore, in order to achieve high spatial resolution, it is required to capture the spectrum at a point having the strongest intensity of excitation light, thus obtaining the best spatial resolution and the optimal spectrum detection capacity. As shown in FIG. 1, all within BB′ zone (a zone in which slope difference in respect to zero-cross point is not more than 10%) in the vicinity of the focus O, the existing confocal microscopy can excite the Raman spectrum of the sample and can be detected by spectrum detection system behind the pinhole. Therefore, the actual detection position of the confocal Raman microscopy is usually placed in out-of-focus BA and A′B′ zones in confocal curve, causing the size of actually detected “micro-area” to be much greater than that of the focus O. Meanwhile, the signal noise ratio of confocal positioning of the utilization of Raman spectrum technology is low. The energy of the Raman spectrum may be further reduced due to the blocking effect of the pinhole; on the other hand, the expansion of the pinhole size for increasing the passing through rate of the spectrum may increase FWHM (full width at half maximum) of confocal axial intensity curve and reduce its positioning accuracy. The size of confocal pinhole in the existing confocal Raman system usually ranges from 150 μm to 200 μm. The relatively large size of pinhole also can't act well on focusing. Above reasons restrict the ability of the confocal Raman microscopy system to detect the micro-area spectrum, constraining its application to a more precise micro-area spectral measurement and analysis area. Therefore, the improvement of focalization precision of the system is the key to improve the spatial resolution.
Kimberley F et al., in “Description and Theory of a Fiber-Optic Confocal and Super-Focal Raman Microspectrometer” in 1996, proposed a method of replacing the pinhole of confocal Raman microscopy with fibre bundle to implement a non-mechanical adjustment of “pinhole” size, in which the spectral resolution of the system does not reduce when the “pinhole” size is increased; E Kenwood Blvd et al., in “Very efficient fluorescent background suppression in confocal Raman microscopy Department of Physics” in 2007, proposed that the fluorescence background of the measured sample could be reduced by about 3 orders of magnitude by combining a picosecond laser of 3-4 ps with the corresponding instantaneous exposure technology, improving the resolution of confocal Raman microscopy; N. Everall et al., in “The Influence of Out-of-Focus Sample Regions on the Surface Specificity of Confocal Raman Microscopy” in 2008, pointed out that a higher axial resolution and signal-to-noise ratio than traditional Confocal Raman spectrometer could be obtained by using an oil-immersion objective with a high numerical aperture (NA=1.4), but this method needs to prepare a sheet for the sample and can't achieve non-contact and non-destructive measurement, thereby restricting the application range of the system; M. J. Pelletier and Neil j. Everall et al., in “Control of the Out-of-Focus Light Intensity in Confocal Raman microscopy using optical preprocessing” in 2009, proposed that an interference of the Raman scattering spectrum intensity at the out-of-focus position could be eliminated by using structure pupil mask or correcting lens, thereby improving the efficiency of the spectrum detection and greatly reducing the influence of the out-of-focus area Raman spectrum in the confocal Raman system on its effective depth resolution.
The above research mainly concentrated on light source system, spectrum detection system, focusing objective system, spectral information processing, etc., which are involved in the confocal Raman microscopy system. While the overall performance of spectrum system could be improved, the spatial resolution of confocal Raman spectroscopy system has not been improved significantly. Therefore, the improvement of the spatial resolution of Raman spectrum system is still a pending issue.
In many research fields such as physical chemistry, biological medicine, film and drug, some further information of the sample could often be obtained in the form of image at the time of analyzing the chemical composition, spatial distribution and physical and chemical properties of the surface of the sample. Thus, there is a requirement to extend Raman spectrum detection from a single point analysis way to the detection and analysis of the sample within a certain area, namely the Raman spectral imaging. However, in order to obtain more accurate and more abundant measurement information, the Raman spectral imaging not only performs multiple-point Raman spectrum detection to the sample, but also needs a relative long time Raman spectrum detection to each point of the multiple-point. As a result, the Raman spectral imaging needs a relative long time for detection. It often takes a few hours to complete imaging. However, in the long-time imaging process, the instrument may be significantly influenced by, for example, environment temperature, vibration, air fluctuation, which may easily cause the instrument system to drift, thereby resulting in the out-of-focus of the detected position of the sample; since the existing confocal Raman spectroscopy does not have the ability of a real-time focus-tracking, the out-of-focus error of the sample which is caused by the offset of detected position can not be compensated, thereby restricting the improvement of the spatial resolution of the confocal Raman spectral imaging technology.
The confocal Raman spectroscopy has varied requirements for the size of detecting converging spot according to the research fields such as the detection of drug, gemstone identification, oil and gas exploration, chemical analysis and archaeology. However, the existing confocal Raman detection technology could not accurately control the size of the converging spot. As a result, the application of the confocal Raman spectral imaging technology in various fields may be constrained.
In the existing confocal Raman spectrometer, the Raman scattering light included in the scattering light of the sample collected by the system is extremely weak, which is only as much as 10−3-10−6 times the Rayleigh light included in the scattering light of the sample collected by the system. Therefore, how to make use of the Rayleigh light in the confocal Raman spectrum detection, which is abandoned in the existing spectrum detection system and which is 103-106 times stronger than the Raman scattering light, to assist the detection is a new approach to improve the spatial resolution of confocal Raman spectroscopy.
On the basis of the above situation, the present invention provides a differential confocal detection system utilizing the Rayleigh light, which is abandoned in the scattering light of the sample collected by the existing confocal Raman spectrum detection system and which is 103-106 times stronger than the Raman scattering light of the sample, to perform highly precise detection. The differential confocal detection system may be combined with the spectrum detection system to synchronously detect the spatial position and spectral information. Therefore, it is desirable to achieve a “mapping & spectrum in one” differential confocal spectral imaging and detection with high spatial resolution and controllable size of measuring converging spot. The achievement of spectrum detection with high spatial resolution is one problem to be solved in the spectral microscopic test field and is of great theoretical and academic value.
The main concept of the present invention is: combining the laser differential confocal technology and spectrum detection technology, the differential confocal system using the Rayleigh light in the scattering light of the sample collected by the system to real-time focus-tracking and detect spatial position, the spectrum detection system using the Raman scattering light in the scattering light of the sample collected by the system to perform spectrum detection, and then fusing the signals of the differential confocal detection system and the Raman spectrum detection system, thereby accomplishing the focus-tracking detection and spot size controllable detection of the laser differential confocal Raman spectroscopy system, namely accomplishing the Raman spectrum detection with high spatial resolution.